Structure-based engineering of benzalacetone synthase

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 17, 2010, Pages 5099-5103

  Shimokawa, Yoshihiko ^a   Morita, Hiroyuki ^a   Abe, Ikuro ^a  

^a UNIVERSITY OF TOKYO   (Japan)

Author keywords

Benzalacetone synthase; Enzyme; Enzyme engineering; Polyketide synthase; Site directed mutagenesis

Indexed keywords

ALANINE; BENZALACETONE SYNTHASE; BENZYLIDENEACETONE; CHALCONE; CHALCONE SYNTHASE; CYSTEINE; MALONYL COENZYME A; NARINGENIN; SERINE; SYNTHETASE; UNCLASSIFIED DRUG;

AMINO ACID SUBSTITUTION; ARTICLE; ENZYME ACTIVITY; ENZYME STRUCTURE; NONHUMAN; NUCLEOTIDE SEQUENCE; POINT MUTATION; PROTEIN EXPRESSION; RHUBARB; X RAY CRYSTALLOGRAPHY;

AMINO ACID SEQUENCE; BASE SEQUENCE; DNA PRIMERS; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; POINT MUTATION; POLYKETIDE SYNTHASES; PROTEIN CONFORMATION; PROTEIN ENGINEERING; PROTEIN FOLDING; SEQUENCE HOMOLOGY, AMINO ACID;

MEDICAGO SATIVA; RHEUM PALMATUM;

EID: 77955661054     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.07.022     Document Type: Article

References (25)

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18. I. Abe, T. Watanabe, H. Morita, T. Kohno, and H. Noguchi Org. Lett. 8 2006 499
19. Site-directed mutagenesis: The plasmids expressing the BAS mutants (L132G, L132A, L132T, L132S, L132C, L132F, L132Y, L132W, and L132P) were constructed with a QuikChange Site-Directed Mutagenesis Kit (Stratagene), according to the manufacturer's protocol, using the following pairs of primers (mutated codons are underlined): L132G (5′-CTCATCGTGTGTTGCGGAGCCGGCGTTGAC- 3′ and 5′-GTCAACGCCGGCTCCGCAACACACGA-3′), L132A (5′-CTCATCGTGTGTTGCGCAGCCGGCGTTGAC-3′ and 5′- CATGTCAACGCCGGCTGCGCAACACACGAT-3′), L132T (5′- CTCATCGTGTGTTGCACAGCCGGCGTTGAC-3′ and 5′-CATGTC AACGCCGGCTGTGCAACACACGAT-3′), L132S (5′- CTCATCGTGTGTTGCTCAGCCGGCGTTGAC-3′ and 5′- GTCAACGCCGGCTGAGCAACACACGATGAG-3′), L132C (5′- CTCATCGTGTGTTGCTGTGCCGGCGTTGAC-3′ and 5′-GTCAAC GCCGGCACAGCAACACACGATGAG-3′), L132F (5′- CTCATCGTGTGTTGCTTTGCCGGCGTTGAC-3′ and 5′- GTCAACGCCGGCAAAGCAACACACGATGAG-3′), L132Y (5′- CTCATCGTGTGTTGCTATGCCGGCGTTGAC-3′ and 5′-GTCAAC GCCGGCATAGCAACACACGATGAG-3′), L132W (5′- CTCATCGTGTGTTGCTGGGCCGGCGTTGAC-3′ and 5′- GTCAACGCCGGCCCAGCAACACACGATGAG-3′), and L132P (5′- CTCATCGTGTGTTGCCCAGCCGGCGTTGAC-3′ and 5′- GTCAACGCCGGCTGGGCAACACACGATGAG-3′). The expression plasmid for the L132T/I214L/L215F triple mutant was also constructed in a similar manner.
20. 600 of 0.6 in LB medium containing 100 μg/mL of ampicillin at 37 °C. Subsequently, 1.0 mM isopropylthio-β-d- galactopyranoside was added to induce protein expression, and the cells were further cultured at 23 °C for 16 h. All of the following procedures were performed at 4 °C. The E. coli cells were harvested by centrifugation at 5000g, and were resuspended in 50 mM Tris-HCl buffer (pH 8.0), containing 0.2 M NaCl, 5% (v/v) glycerol and 2 mM DTT (buffer A). The cells were disrupted by sonication, and the lysate was centrifuged at 12,000g for 30 min. The supernatant was loaded onto a Glutathione Sepharose 4B affinity column (GE Healthcare) equilibrated with buffer A, and the column was then washed with buffer A. The GST-tag was cleaved on the column by PreScission Protease (GE Healthcare) overnight, and then the recombinant BAS mutant was eluted with buffer A. The resultant BAS protein thus contains three additional residues (Gly-Pro-Gly) at the N-terminal flanking region, derived from the PreScission Protease recognition sequence. The protein concentration was determined by the Bradford method (Protein Assay, Bio-Rad) with bovine serum albumin as the standard.
21. 2O and MeOH, both containing 0.1% TFA: 0-5 min, 30% MeOH; 5-17 min, linear gradient from 30% to 60% MeOH; 17-25 min, 60% MeOH; 25-27 min, linear gradient from 60% to 70% MeOH. Elutions were monitored by a multichannel UV detector (MULTI 340, JASCO) at 280; UV spectra (198-400 nm) were recorded every 0.4 s. Online LC-ESIMS spectra were measured with an Agilent Technologies HPLC 1100 series HPLC coupled to a Bruker Daltonics Esquire4000 ion trap mass spectrometer fitted with an ESI source. HPLC separations were performed under the same conditions as described above. The ESI capillary temperature and the capillary voltage were 320 °C and 4.0 V, respectively. The tube lens offset was set at 20.0 V. All spectra were obtained in the positive mode, over a mass range of m/z 50-500, and at a range of one scan every 0.2 s. The collision gas was helium, and the relative collision energy scale was set at 30.0% (1.5 eV).
22. 14C]Malonyl-CoA was purchased from GE Healthcare. Benzoyl-CoA, phenylacetyl-CoA and malonyl-CoA were purchased from Sigma
23. cat values (average of triplicates), using the EnzFitter software (BIOSOFT).
24. H. Morita, Y. Takahashi, H. Noguchi, and I. Abe Biochem. Biophys. Res. Commun. 279 2000 190
25. Homology modeling: The models of the BAS L132T and L132W were generated by the swiss-model package (http://expasy.ch/spdbv/ ) provided by the Swiss-PDB-Viewer program (Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714) based on the crystal structure of wild-type BAS (PDB code: 3A5Q ). The model quality was assessed using PROCHECK (Liang, J.; Edelsbrunner, H.; Woodward, C. Protein Sci. 1998, 7, 1884). In the Ramachandran plot calculated for the model, most of the amino acid residues were present in the energetically allowed regions with only a few exceptions, primarily Gly residues that can adopt phi/psi angles in all four quadrants. The cavity volume was calculated by the program castp (http://cast.engr.uic.edu/cast/ ). All protein structure figures were prepared with pymol (DeLano Scientific, http://www.pymol.org ).